PII:S0273-1223(96)00146-1

8) Pergamon War. Sci. Tech. Vol. 32. No. 12. pp. 121-129. 1995.Copyright Cl 1996 lAWQ. Published by Elsevier Science Ltd

Printed io Great Britain . All rights reserved .0273-1223195 $9 '50 +0-00

EFFECT OF TRACE METALS ON THEANAEROBIC DEGRADATION OFVOLATILE FATTY ACIDS IN MOLASSESSTILLAGE

A. Espinosa, L. Rosas, K. IIangovan and A. Noyola

Instituto de Ingenieria; UNAM, Apdo. Postal70-472. Coyoacan 04510. Mexico D.F.,Mexico

ABSTRACT

A laboratory UASB reactor was fed with cane molasses stillage at organic loadings from 5 to 21.5 kgCOD/m3 d, With an organic load of 17.4 kg COD/m3 d. an accumulationof VFA.principally propionicacid.was observed due to little bioavailabilityor lack of trace metals (Fe. Ni, Co and Mol. Associated to this. theperformance of the UASB reactor was low (44% COD removal efficiency). with an alkalinity ratio above0.4. The addition of Fe (lOO mgll). Ni (15 mg/l). Co (10 mg/l) and Mo (0.2 mgll) to the influent reducedsignificantly the level of propionic acid (5291mg/lto 251 mg/l) and acetic acid (1100 mgll to 158mgll). TheCOD removal efficiency increased from 44% to 58%. the biogasproduction from 10.7 to 14.8 Vd(NTP) and0.085 to 0.32 g CH4-COD/g SSV d for specific sludge metbanogenic activity with propionic acid assubstrate. These improved results were obtained with high COD (68.9 gil) and organic load (21.5 kgCOD/m3 d). Copyrighte 1996IAWQ. Publishedby ElsevierScienceLtd.

KEYWORDS

Anaerobic digestion: VASB reactor; molasses stillage: vinasse: trace metals: volatile fatty acids: alkalinityratio.

INTRODVcnON

Molasses stillage, also known as vinasse, is a liquid waste from alcohol production by fermentation of caneor beet molasses. Besides a low pH and high temperature, cane molasses stillage has high amounts oforganic matter, potassium, sulphates and chlorides making it complicated for wastewater treatment process.A common situation encountered during anaerobic treatment of this waste is the volatile fatty acid (VFA)accumulation, principally propionic acid (Sanchez Riera et al., 1985; Tielbaard, 1992). Propionic acidcontributed with 70-90% to the COD of VFA in a thermophilic VASB reactor fed with beet molassesstilIage (Wiegant et al., 1985). In an anaerobic filter fed with cane molasses stillage, the concentration ofPropionic acid was' 1.2 gil (Bories et al., 1988). On the other hand, Vlissidis and Zouboulis (1993) reportedthat typical composition of the effluent, from an anaerobic contact reactor treating beet molasses stillage.Was 78% acetic, 6% propionic. 12% butyric acids and 4% of longer chain volatile fatty acids. In spite of?igh concentrations of propionic acid (over 1 gil) that may be considered inhibitory, the anaerobic reactorsIn those experiments were operated steadily. The complex characteristics of cane molasses stillage limit thedegradation of this intermediate substrate, while endogenously producing enough alkalinity to buffer the

121

122 A.ESPINOSA et al.

liquor. The main inhibitory inorganic constituents are potassium (4-10 gil), sulphates (3-6 gil), and in alesser extent, chlorides (3 gil).

Kugelman and McCarty (1965) reported that 5.9 gil of potassium decreased 50% the methanogenic activity.During anaerobic digestion. sulphates are reduced to sulphides, which are the major source of sulphur tomethanogenic bacteria. The optimum level is about 25 mgll and a concentration up to 150 mgll of un­ionized H2S inhibits the methanogenic activity (Speece, 1983). Concentrations of 90 and 250 mgll ofhydrogen sulphide decreased 50 % the methanogenic activity at levels of pH 7.8-8 and 6.4-7.2, respectively(Koster et al., 1986). Additionally, the formation of precipitates. between sulphides and trace metals,reduces their bioavailability to anaerobic microorganism (Callender and Barford, 1983). More recently,Ilangovan and Noyola (1993) investigated that the iron accumulated in the sludge of an UASB reactor fedwith vinasse was determined as sulphides (29%) and carbonate forms (23%).

It has been reported that addition of 7.4 mgll of nickel, to a downflow fixed-film reactor treating a cheesefactory effluent decreased the level of butyric acid from 2300 mgll to 150 mgll (Canovas-Dfaz and Howell,1990). Hoban and van den Berg (1979) found that the addition of 20 mrnol/l of ferrous chloride increasedthe conversion of acetic acid to methane. The addition of iron (615 mgll) to a tower fermenter fed withvinasse from cane juice, allowed the increase of space loadings from 0.8 to 5.3 kg COD/m3 d with lowlevels of VFA. The presence of nickel (100 nM) and cobalt (50 nM) or in combination of both elementsincreased the conversion of acetic acid to methane (Murray and van den Berg, 1981).

Cane molasses stillage lacks molybdenum, cobalt and only has small amounts of nickel (Ilangovan andNoyola, 1993), so VFA accumulation during anaerobic treatment of molasses stillage may be due to thisdeficiency, among other limitations set by the complex characteristics of this wastewater. The objective ofthis research was to reduce the levels of VFA, mainly propionic acid, with the addition of Fe, Ni, Co and Moto a laboratory UASB reactor fed with cane molasses stillage at high space loadings (over 17 kg COD/m3 d),in order to increase its COD removal efficiency.

MATERIALS AND METHODS

The molasses stillage (vinasse) was anaerobically treated in a glass made UASB (Upflow Anaerobic SludgeBlanket) reactor (volume 2.3 1;internal diameter 8 ern) at an average hydraulic retention time (8) of 3.5 daysunder controlled temperature (35°C ± I). The wastewater was continuously fed to the bottom of the reactorwith a peristaltic pump; the reactor was seeded with an adapted anaerobic sludge. The biogas produced waspurified in a separate glass column, which contained a solution of Fe-EDTA. The biogas from the UASBwas continuously introduced to the column bottom and after the removal of H2S. recirculated to the bottomof the UASB reactor. In this arrangement, the H2S-free biogas was used in order to remove by stripping theH2S dissolved in the UASB liquor (Noyola et al., 1991).

Physicochemical characteristics (COD, pH, solids. sulphides and sulphates) of molasses stillage were carriedout following Standard Methods (APHA, 1990). The biogas composition was determined by gaschromatography using a thermal conductivity detector, a Porapak Q and 5 A molecular sieve columns.Volatile fatty acids were measured using a gas chromatograph with a flame ionization detector using fusedsilica column (superox-FA). The concentration of metals (Fe, Ni, Mo, Co and K) was determined by atomicabsorption using an atomic absorption spectrometer model llOOB (Perkin Elmer, West Germany). The metalspeciation on sludge reactor was carried out following Stover et al. (1976). The wastewater samples(influent and effluent) were diluted 20 times and 24 times for sludge samples. Under these circumstances thedetection limits of the spectrometer were 0.12 mgll (Co), 0.08 mgll (Ni) and 0.6 mgll (Mo), Formethanogenic activity measurements, the mineral medium was prepared and inoculation was realizedaccording to Hungate (1969) and Balch et aL (1979). The alkalinity was determined by potentiometrictitration at pH 5.75 and 4.3. The bicarbonate alkalinity was determined at pH 5.75 (Jenkins et al., 1991) andthe total alkalinity at pH 4.3. The ratio between them (a = (alk 4.3-alk 5.75)/alk 4.3) was used as a controlparameter for operation of the UASB reactor. For a good balance in the reactor the ratio should be keptbelow 0.4 following this criteria, when a was above 0.4. around 2 gil of NaHC03 was added.

Trace metals and volatile (atty acids 123

The experimentationwas divided into 4 periods. In the first one. the reactor was fed with several dilutions ofmolasses stillage. For the second period. the reactor was fed at space loading of 17.4 kg COD/m 3 d withmolasses stillage diluted to 70% with tap water. For the third and fourth periods. the new sample ofwastewater had a higher COD strength and therefore it was diluted to 60% vinasse, In the third period. thereactor was fed at 18.8 kg CODtm3 d. On day 298. the addition of 100 mg/l of iron. 15 mg/l of nickel. 10mgll of cobalt and 0.2 mgll of molybdenum marked the beginningof the fourth period. These concentrationswerechosen accordingly to Weiland and Rozzi (1991).

RESULTS

During the first period. the UASB reactor was operated at different space loadings with various fractions ofmolasses stillage (20. 50 and 60% vinasse). At a fraction of 60% vinasse and a space load of 14.5 kgCODtm 3 d. the reactor still achieved high COO removal and good stability (Table 1). Under theseconditions. only 142 mgll of acetic and 29 mgll of propionic acids were detected in the UASB effluent andthe ratio of alkalinity was lower than 0.3.

Table 1. Operationalconditions for UASBreactor without VFA accumulation (period I)

Vinasse CODt 9 Spaceload,Bv Gas production Removal Total alkalinity AlkalinityFraction (mg/l) (days) (kg COD/mJ d) (INTPI d) efficiency (mgCaCOJ!I) ratio (a)(010) CODt (%)

20 16862 3.4 5 2.1 73 2424 0.2850 37868 3.4 11 5.4 73 6766 0.2260 49152 3.4 14.5 9.7 71 8895 0.23

After 63 days of operation. the second period started with the increase of space load to 17.4 kg CODtm3 dby feeding 70% of molasses stillage and an accumulation of acetic (807 mg/l) and propionic acids (4095mg/l) was observed (Fig. 1). The ratio of alkalinity in the effluent increased over 0.4 (Fig. 2) due to theproductionof VFA alkalinity.

From the results. 60% of the mean VFA alkalinity. difference between alkalinities at pH 5.75 and 4.3 (4451mgll as CaC03). was due to propionicacid (4095 mg/l) accumulation (Table 2). Additionally.84% ofVFA­COD and 19%of soluble COD (COOs) were given by propionicacid.

To start the third period. on day 215. the space loading was increased to 18.8 kg CODtm 3 d and the ratio ofalkalinityrose near 0.5 (Fig. 2). Apparently. this change was due to an increase in the levels of propionicandvalerie acids (Fig. I. Table 2). Twenty two days later (day 237). the ratio of alkalinity in the reactor reacheda steady value (Fig. 2) with an average of 0.49. In this period. the propionic acid (5291 mgll) contributedwith 52%. 77% and 24% to VFA alkalinity. VFA-eOD and soluble COD. respectively. In spite of the highlevels of VFA and alkalinity ratio. the stability of the UASB reactor was not affected. Some externalalkalinity was necessary in the second and third period. when eventually around 2 gil of sodium bicarbonatewas used.

The soluble COD removal. on the second and third periods, was low (40% and 42%) although the biogasProduction averaged 14.5 litres (NTP) per litre of vinasse fed. The removal of sulphates was 88% and 93%.with 145 and 121 mgll of total sulphides in the effluent, respectively (Table 2). Analysis of trace metals inthe molasses stillage (Table 2), showed absence of nickel, cobalt and molybdenum. Results of metalspeciation in the UASB sludge showed presenceof iron in all forms (Table 3).

On day 298. the fourth period started with the addition of iron (100 mg/l). nickel (15 mg/l), cobalt (10 mg/l)and molybdenum (0.2 mg/l) to the feeding solution (60% vinasse). Twelve days later (day 310). the ratio ofa1kaIinity decreased from 0.51 to 0.34 and propionic acid from 5210 to 626 mg/l (Figs I and 2). Thereductionaverage was 39% for alkalinity ratio. 86% for acetic acid. 95% for propionic acid and near 100%for valerie acid (Table 2). Under such operating conditions, the reactor developed a good capacity to

124 A. ESPINOSA et al.

neutralize the low influent plf, increasing it from 4.72 to 7.79 in the effluent, without the need of externalalkalinity.

As an expected consequence, the supplementation of trace metals resulted in an increase of 32% in the CODremoval efficiency and 38% in gas production. The removal of sulphates and the methane yield were notaffected by metal addition. However. the methanogenic activity of the sludge was increased by the presenceof metals. In such tests. with propionic acid as substrate. the methanogenic activity increased from 0.085 to0.32 g CH4-COD/g VSS d. and with acetic acid from 0.23 to 0.32 g CH4-COD/g VSS d.

Table 2 shows that around 80% of added metals were kept by the sludge. In Table 3, it can be noticed thatthe carbonate and sulphide forms of iron increased markedly during period 4. Nickel and cobalt were finallyfound in the sludge. mainly precipitated as sulphides and carbonates; molybdenum was not detected.

Table 2. Physicochemical characteristics and operational conditions of the UASB reactor treating molassesstillage at different periods of operation

Parameten Units Period 1 Period 2 Period 3 Period 4(Days 1-63) (Days 64-214) (Days 215-297) (Days 298-407)

Influent Effluent Influent Effiuent Influent Effiuent Influent EffiuentpH 3.83 7.61 418 7.56 5.2 7.71 4.72 7.79Alk.5.75 mgll csco, 6826 6534 7164 9503A1k.4.3 mgll CaCO] 8895 10985 13966 12438Alkalinity ratio a 0.23 0.40 0.49 0.30Total COD mgll 49152 15119 61125 34191 63862 36504 68907 28574Soluble COD mgll 42351 14181 53525 32110 58286 33709 61770 27130Acetic mgll 142 807 1100 158Propionic mgll 29 4095 5291 251Butyric mgll n. d. 85 44 n.d.n-valeric mgll n. d. 27 364 n.d.Iso-valerie mgll n.d. 63 154 7Sulphates mgll 1690 470 2850 310 1970 130 1640 200Sulphides mgll 61 145 121 91Potassium mgll 5886 6214 7844 9257 4611 5817 4484 5721Iron mgll 41 32 50 15 39 II 154 24Nickel mgll n. d. n. d. n.d. n. d. n. d. n. d. 15- 4Cobalt mgll n. d. n. d. n. d. n. d. n. d. n. d. 10- 1.7Molybdenum mgll n.d n d n. d. n. d. n. d. n. d. 0.2- n. d.Gas Production NTP (I1l.d) 4.20 380 4.65 6.43Methane % 50 46 53 56Carbon dioxide % 50 54 47 44Methane yield I CHJg CODrem 0.21 0.22 0.30 0.29COD t removal % 71 44 44 58COD s removal % 71 40 42 56VSS removal % 62 55 68 76Space load kgCOD/m] d 14.5 17.4 18.8 21.5HRT(8) days 3.4 3.5 3.4 3.2

n. d.; not detected (for detection limits, see Materials and Methods). - Added concentration, not analyzed

Tracemetalsand volatile fatty acids 125

Table 3. Metal speciation in UASB sludge (mglg TSS)

Metal speciation Periods Iron Nickel Cobalt MolybdenumExchangeable 2 0.30 n. d. n. d. n. d.

3 0.21 n. d n d. n. d.4 068 0.05 0.16 n. d.

Sorbed 2 0.19 n. d n. d. n. d.3 0.15 n. d n. d. n. d.4 0.22 0.07 0.10 n. d.

Organically 2 0.90 n. d n. d. n. d.bound 3 0.53 n. d n. d. n. d.

4 0.67 0.08 0.12 n. d.Carbonate 2 0.20 n. d n. d. n. d.

3 0.25 n. d n. d. n. d.4 3.59 0.51 0.21 n. d.

Sulphide 2 0.20 n.d n. d. n. d.3 023 n. d n. d. n. d.4 2.26 0.61 038 n. d.

n.d.; not detected (for detection limits, see Materials and Methods) .

DISCUSSION

During the first period of operation, when the UASB reactor was fed at space loads below 14.5 kgCOD/m3 d with diluted vinasse (20, 50 and 60% vinasse: 16.8,37.8,49.1 gil COD), the COD removal wasabout 72%, the alkalinity ratio was lower than 0.3 and VFAs were lower than 0.5 gil (Table I) . However,when the new sample of wastewater was diluted to 70% and 60% vinasse for a COD of 6I.l and 63.8 gil(second and third period, space load 17 kg COD/m3 d), the COD removal efficiency dropped to 44% (Table2) and the alkalinity ratio went above 0.4 due to an internal generation of VFA (Figs I and 2). The sameeffect of vinasse dilution also was observed in an anaerobic filter reactor; with a COD of 40 and 60 gil, theCOD removal efficiencies were above 60%, but for 80 gil COD it dropped to 50% (Silverio et al., 1986). Inthe same way, Tielbaard (1992) recommended that the vinasse should be diluted to a COD of 30 gil andapplied to a volumetric loading rate of 16 kg COD/m3 d to obtain a COD removal efficiency in the 56-74%range.

On the other hand, VFA accumulation was observed when undiluted molasses stillage was fed to anaerobicprocess (Sanchez Riera et al., 1985; Vlissidis and Zouboulis, 1993) or when high organic loads were appliedIWeigant et al., 1985; Bories et al., 1988). Although the acetic and valerie acids were found in the effluentof UASB reactor, the levels of propionic acid predominated (Fig. I, Table 2). The results obtained are in fullOr partial agreement with the reports of prior studies (S~chez Riera et al., 1985; Wiegant et al., 1985;Bories et al., 1988). In this study, in spite of high concentration of propionic acid (5291 mgll) and alkalinityratio (0.49) the stability of the reactor was not adversely affected.

Inhibition of the COD removal efficiencies and the VFA accumulation could be related to the presence ofoxidized phenolic compounds (Wiegant et al., 1985) and caramelized components (Tielbaard, 1992).Nevertheless, the lack of trace metals (Ni, Co, Mo) in the wastewater was evident and it might be at theorigin of the low performance of the UASB reactor at high organic loads. Before the supplementation oftrace metals, 39 to 50 mgll of iron were detected in the vinasse (Table 2). Also, the speciation of iron in theslUdge (Table 3) showed that 0.3 and 0.21 mg of iron per gram TSS (periods 2 and 3) were found inexchangeable form, available to the bacterial population. However, the bioavailability of iron could bereduced by the natural chelators that bind the metals tightly, so they are not bioavailabIe. Additionally, highlevels of potassium in the vinasse could also have a negative influence on the availability of essentialrnicronutrients (Ilangovan and Noyola, 1993).

126

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O ~----~------------r------r------------...,

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'""0U..u 4- -'c0Q.0

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o lLJ_-~,-"1L-_'--__--J -l-,,-_--'-__-l

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Co 400E

200

60 100

, Bu ty ric ac re I .j

250

T llTlo (dey.'

rso -valerrc ac id (- -)

400 460

Figure I. Evolution of volatile fatly acids in UASB reacto r.

Trace metals and volatile rattyacids 127

c> ._ ,-- - - - .--- -------------r------,-- - - - - --- - - - -..,

Period 1 Per iod 2 Per iod 3 Per iod 4

4504003503 0 02502 0 01501005 0

o l- '_'-_~'__ _ _''_____'_l___'_ L1. ___'_ ___' ___'

o

c> --'

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00

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'" 40:0

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0.

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iii 5

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Figure 2. Alkalinity ratio. COOs removal and biogas production in VASB reactor.

128 A.ESPINOSA et al:

Addition of trace elements. iron included, to the UASB reactor along with the influent significantly reducedthe VFA concentration (Fig. 1), increasing the overall performance of the UASB reactor and the specificmethanogenic activity of the sludge, mainly with propionate as substrate. Speece (1988) reported thatalthough the soluble iron. cobalt and nickel concentrations appeared to be ample on some sludge digesters,their supplementation caused a stimulation in the gas production. Apparently, considering the accumulationof VFA. the lack of trace metals affected the performance of bacterial populations of Obligate HydrogenProducing Acetogens (OHPA), hydrogen-utilizing methanogens and methanogens converting acetate tomethane. It has been reported that the addition of iron increased the activity and growth of the hydrogen­oxidizing methanogens (Patel et al., 1978) and the bacteria converting acetic acid to methane (Hoban andvan den Berg, 1979). On the other hand, nickel is a component of the factor F430 from methanogenicbacteria (Dickert et al., 1981). This factor is contained in the methyl-CoM methyl reductase that participatesin the terminal step of methanogenesis. Molybdenum is associated with a molybdenum protein that reducesCO2 to formate and cobalt could be associated with the formation of corrinoid compounds (Schonheit etal., 1979) and of vitamin B12 involved in methyl group transfer.

As a result of metal addition, the UASB reactor was able to receive higher COD (68.9 gil) and space loadingrate (21.5 kg COD/m 3 d) than those recommended by Tielbaard (1992) for the same kind of wastewater anddeveloped the ability to produce a high buffer capacity that neutralized the low influent pH. The complexcharacteristics of vinasse, with high content of COD and inorganic salts, enables the reactor to produceenough bicarbonate ions during the conversion of substrate to methane, increasing the influent pH (below 5)to an effluent pH above 7.5. In this case, the self-regulation of the anaerobic digestion brings importanteconomic consequences due to the savings in neutralizing reagents.

CONCLUSIONS

The accumulation of VFA in a UASB reactor fed with vinasse at high organic loads. over 17 kg COD/m 3 d,was due to a total or partial lack of trace metals (Fe. Ni, Co and Mo). The addition of such metals to theinfluent reduced the VFA concentration by 94% (95% for propionic acid), increased 32% the COD removalefficiency, 38% the biogas production and the specific sludge methanogenic activity (39% and 276% withacetic and propionic acids as substrates respectively). This operational improvement allowed an influentCOD concentration of 68.9 gil and an organic loading of 21.5 kg COD/m 3 d with low VFA concentration inthe effluent and good reactor stability. The alkalinity ratio (ex) was a good parameter that reflected quicklyenough the variations in the concentration of VFA in the reactor, and may be used as a simple andinexpensive measure of reactor stability.

ACKNOWLEDGMENTS

The authors thank DGAPA-UNAM (Direcci6n General de Asuntos para el Personal Academico-UniversldadNacional Aut6noma de Mexico) for the financial assistance (projects IN 302289 and IN 301392). ArmandoSaules and Claudia Bermudez are gratefully acknowledged for their technical assistance.

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